Hypoplastic Left Heart Syndrome (HLHS) is a congenital heart defect (CHD) characterized by a small left ventricle (LV) and hypoplastic aorta and aortic/mitral valves. A genetic etiology for HLHS is strongly indicated by high recurrence risk, but the genetic underpinning for HLHS is poorly understood. Clinical studies suggest HLHS is multigenic and genetically heterogeneous. Insights into the genetics of HLHS has come from our recent recovery of the first mouse models of HLHS from a large-scale mouse mutagenesis screen. From 8 independent HLHS mouse lines recovered, 330 mutations were identified, with no genes shared in common between the 8 lines. These findings indicate HLHS is profoundly genetically heterogeneous, consistent with the human studies. Detailed analysis of one mutant mouse line, Ohia, showed HLSH is elicited by mutations in two genes: Sap130, a Sin3a associated protein in the chromatin modifying histone deacetylase complex (HDAC), and Pcdha9, a protocadherin mediating cell-cell adhesion. The LV hypoplasia was shown to be elicited by the Sap130 mutation, a finding confirmed with replication of a small ventricle phenotype in a CRISPR generated sap130a zebrafish mutant. The LV hypoplasia was associated with a cardiomyocyte cell proliferation defect and cardiomyocyte cell cycle arrest. In this study, we will investigate the cellular and molecular mechanisms and genetic interactions driving the LV hypoplasia in HLHS, leveraging the unique strengths of the zebrafish and mouse models. In Aim 1, we will employ lineage tracing studies in zebrafish and experiments with Cre deletion of Sap130 in mice to test the hypothesis that Sap130 functions in a cell autonomous manner to regulate ventricular/LV growth. These studies will delineate the cellular context in which Sap130 regulates LV growth. In Aim 2, we will investigate the hypothesis that the hypomorphic Sap130Ohia mutation causes LV hypoplasia via target genes that regulate cardiomyocyte cell cycle and cell proliferation. These studies will focus on Meis1, a Sap130 target gene, also known to regulate cardiomyocyte cell cycle and postnatal cell cycle arrest. In parallel, additional candidate genes identified via Sap130 ChIP-seq and RNA-seq analysis will be assessed for their role in LV hypoplasia with production and analysis of CRISPR targeted embryos and mice. In Aim 3, we will probe the interaction of chromatin modifiers with the Ras/MAPK signaling pathway in the pathogenesis of HLHS using antisense morpholino gene knockdown in zebrafish with a sensitized genetic background. Positive genetic interactions will be validated using mutant or CRISPR targeted zebrafish or mice. This study is motivated by the unexpected recovery of mutations in chromatin modifiers and Ras/MAPK pathway components in all 8 HLHS mouse lines, suggesting chromatin modifiers in combination with dysregulated Ras/MAPK signaling may contribute to the LV hypoplasia and complex genetics of HLHS. Together these studies will help to elucidate the cellular and molecular mechanisms driving the ventricular hypoplasia in HLHS, findings that may yield new therapeutic targets for fetal intervention to recover LV growth. !